Osteoporosis is a major health problem due, in part, to low bone mass. The fundamental goal of the applicants' research is to find mechanisms for increasing bone mass in the adult skeleton. The purpose of this project is to identify early response genes for mechanically-induced bone formation and examine the association of these genes with first messengers, such as prostaglandins and nitric oxide. This project will use an in vivo model of controlled external loading. The hypotheses to be tested are: (1) in vivo loading changes mRNA levels of up to 7 early response genes that are indomethacin-sensitive and bone-specific; (2) these gene responses are first detectable at 30 minutes after the initial load, and indomethacin prevents the response for up to 8 hours while blocking bone adaptation to repeated loading; (3) nitric oxide is a biochemical messenger of mechanical loading and stimulates the same bone-specific early response genes that indomethacin suppresses; and (4) indomethacin-sensitive, early response genes for loading are not stimulated by other bone-forming agents, such as parathyroid hormone and growth hormone. The Specific Aims are: (1) to identify, by ddPCR, a subset of periosteal genes, with mRNA levels elevated or suppressed 30 minutes after loading, that are sensitive to indomethacin and specific to loaded bone; (2) to determine indomethacin effects on the time course (0.25-8.0 hrs) of mRNA levels of these genes by Northern blots and associate the differences with histomorphometric measurements at 3 weeks of loading; (3) to compare differences in loading response for these genes, between indomethacin and L-NG-monomethyl-arginine (L-NMMA) treatment at 30 minutes, 2 hours, and 3 weeks; and (4) to compare differences in gene response to loading, parathyroid hormone or growth hormone treatment at 30 minutes, 2 hours and 3 weeks. It is suggested that the results from these studies will identify indomethacin-sensitive loading early response genes and characterize their time course, association with first messengers, and association with other bone-forming agents. This knowledge, from an in vivo model, is intended to contribute to the knowledge gained from in vitro studies, aid in the discovery of bone-specific signal pathways for mechanical stimulation, and may help in the design of treatments to increase bone response to mechanical loading and increase bone mass.